A powerful new technology for gene therapy is now available. The technology uses an integrase enzyme from phage phiC31 that mediates chromosomal integration of therapeutic genes into a limited number of native sequences in mammalian genomes. This innovative technique is the only one that provides efficient site-specific, rather than random, chromosomal integration. The method also provides permanent, strong expression of the therapeutic gene. The integrase system has been validated in animal models in a number of tissues, species, and diseases. The focus of this proposal is to use the phiC31 integrase system to create a novel methodology for safe, simple, and permanent transfer of genes to the retina of larger mammals. We demonstrated an electroporation method that was effective for DNA delivery to rat retina and showed that integrase provided strong, long-term expression of a marker gene in the retinal pigment epithelium. Methods will now be established to achieve efficient gene delivery and site-specific integration into the retina of rabbits, as a model for later dog and human work. Innovative electroporation methods will be developed with collaborators from physics and ophthalmology to achieve surgical placement of electrodes to provide effective and precise delivery of therapeutic DNA to the RPE, with minimal tissue damage. The phiC31 integrase will be utilized to provide site-specific genomic integration and long-term expression of transferred genes in the retina. Delivery parameters will initially be optimized with in vitro model systems, while imaging of luciferase in the rabbit eye will be employed in long-term in vivo studies. To lay the groundwork for studies in the RPE65 dog to follow, site-specific integration into cultured dog cells will be demonstrated and genomic integration hotspots will be characterized. These experiments will move the phiC31 integrase [unreadable] [unreadable] [unreadable]